Biofuel from D-Xylose - the Second Most Abundant Sugar

GENERAL I ARTICLE Biofuel from D-xylose - the Second Most Abundant Sugar

Anil Lachke

In the biosphere we find cellulose and hemicellulose as the major polysaccharides. On acid or enzymatic hydrolysis, D-glucose is produced from cellulose and D-xylose is pro duced from xylans as the major sugar in the hydrolysate. Initially it was believed that yeasts do not fermentD-xylose to ethanol although many are capable of producing xylitol.

Anil Lachke is a scientist Twenty years ago, a few yeasts that could convertD-xylose in the Division of to ethanol were found. Ethanol is viewed as a potential fuel Biochemical Sciences of that is available from biomass and hence new methods to National Chemical generate ethanol from hitherto inaccessible sources are Laboratory, Pune. His major research interests gaining importance. Biotechnology for efficient utilization include pentose metabo- of lignocellulose wastes as fuels relies on the utilization of lism in yeasts, biotechnol both the cellulosic as well as hemicellulosic portions of the ogy for biomass utiliza biomass. In this article, conversion of xylose into ethanol tion and biodeinking for recycling of waste paper. is discussed. He takes special interest in popularization of Henry Ford was once asked about the fate of his automobile science and technology. business if the petroleum supplies of the world run out. He He likes Indian classical exclaimed, "we can get fuel from the sumac by road side or from music, yoga and drawing. weeds, sawdust almost anything. There is fuel in every bit ofvegetable matter that can be fermented .... A nd it remains for someone to find out how this fuel can be produced commercially ... Better fuel at a cheaper price than we now know." The optimism shown by Henry Ford must be appreciated.

Many countries are showing increasing interest in obtaining a large fraction of their energy needs from biomass. More than 25% of the cars in Brazil run on gasohol (Box 1) - a mixture of petrol and alcohol. They produce alcohol from molasses. Cur rent scientific opinion seems to favour the view that the world's Keywords fossil fu~l resources are steadily diminishing. Alternative sources D-xylose, biofuel. of energy must therefore be investigated urgently. There is no

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Box t. Gasohol- An Alternative Fuel

AlcOhol was identified as a possible replacement fuel during 1920's when there was a growing demand for gasoline and potential shortage of oil. Many automobile giants like General Motors and Ford, predicted that alcohol could be a viable fuel for vehicles. A blend of gasoline and alcohol that is referred to as 'gasohol' was tried out in various cars. However, gasohol got a bad name because of excess use or wrong choice of alcohol. Ofthe two types of alcohols that could be used, ethyl alcohol and methyl alcohol can be used by itselfas a fuel but when mixed with gasoline, it is limited to 10% by volume. Methyl alcohol is very corrosive and this limits its use in the mixture.

The heat content of a litre of alcohol is less than gasoline and hence more alcohol is required to achieve the same power levels as that of gasoline. The penalty is decreased fuel economy and lower driving range without installing bigger fuel tanks. Another disadvantage of alcohol as a fuel is its higher volatility. During hot weather driving, the fuel vaporises easily and vapour lock in the engine can occur. This can make the engine run rough or even prevent it from running. An advantage of using alcohol as a fuel is that it mixes easily with water and prevents ice formation in cold weather. It also has a high octane rating than gasoline, which allows engine compression ratios to be increased and ignition timing to be advanced for better performance.

The successes in development of other alternate energy sources may limit the use of alcohol as a fuel, but there has been some work in using alcohol in conjunction with fuel cells to produce power for the future vehicles. Alcohol has been used as a fuel for centuries and it may well be the fuel of the future too. Currently, there is only one major fuel company in Canada, Mohawk, supplying fuel with alcohol in it. Brazil is also supplier and user of gasohol for a number of years. doubt that in the long run we have to tum to renewable sources of food and energy.

Over 150 billion tonnes of organic substances are photosynthe sized annually which consists of three major constituents, namely cellulose, hemicellulose (Box 2) and lignin with an average proportion of 4:3:3. The xylan (hemicellulose) content of the hardwood and the softwood lies in the range of 20-25% and 7-12%, respectively.

The incentives for energy recovery from wastes are most attrac ti ve in the rural areas of India. Lignocellulose as a raw material Over 150 billion for bioconversion has a role in energy production; it can prevent tonnes of organic deterioration of the environment, and facilitate waste manage substances are ment (Figure 2). Large quantities of lignocellulosic materials in photosynthesized the form of agricultural and forest residues are available in annually.

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Box 2. Hemicellulose

Plants contain about 6 x 1011 tonnes of hemicellulose and about 3 x 10 10 tonnes are photosynthesized annually by higher plants to make over one third of their dry weight. The close association of hemicelluloses with cellulose and lignin confer rigidity to the plant cell wall.

The hemicelluloses are composed of a linear as well as branched hetero- and homopolymers of pentosans (D-xylose and L-arabinose) and hexosans (D-glucose, D-mannose and D-galactose). Unlike cellulose hemicelluloses show structural variability (Figure I).

As compared to cellulose, hemicelluloses are low molecular weight polymers with a degree of polymer ization of around 200. Native hemicelluloses like xylan is composed of 85-90% D-xylose and a small amount of L-arabinose and traces of glucuronic acid. HemiceUuloses have more branches and are less crystalline than cellulose. The glycosidic linkages between the anhydro-D-xylose residues in xylan are more susceptible to acid or enzymatic hydrolysis than linkages between anhydro-D-glucose residues in cellulose. As a result, pentose sugars can be obtained readily in hydrolysates with sufficient yields from hemicelluloses. Considering the complex structure of hemicelluloses, a diverse set of enzymes is necessary for its hydrolysis to form soluble pentose sugars. These include: Endo-l, 4-fJ-D-xylanase, Exo- 1,4-p-xylanase, 1,4-fJ-D-xylosidase and a-L-arabinofuranosidase.

Figure 1. @ G) OH OH(j) H~2C HO~OH HO 0 O~rf HO o 0 HO HOH2C HO @ ~HOH2~ D-GLUCOPYRANOSE CELLOBIOSE UNIT RING

CELLULOSE

P(1~ 4)-D-XYLOPYRANOSE LINKAGE OH a-(1~3)- L-ARABINOFURANOSE LINKAGE '" __ O~O~O o 000 O~-\° -0-- HO~~ AcO~ OH H;?O HO OH 0 OH OAc D-XYLOPYRANOSE RING 0

HOOC OH a-(1----. 2) - 4-0-METHYL-D-GLUCURONIC Ac- Acetyl group ACID LINKAGE

MeO HO

XYLAN

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Figure 2.

Xylose Glucose Mannose, Arabinose Galactose

• Energy (ATP) • Fuel • Energy (ATP) • Chemicals • Protein (SCP) • Chemicals • Proteins(SCP) • Solvents • Fermentation • Solvents • Chemicals Products • Beverages • Xylitol • Chemicals • Energy (ATP) • Fructose • Petrochemical Synthesis • Food! Feed

India. For example, India's production of wheat straw and rice straw is over 200 mlnion tonnes per year. Although straw has low protein content it is rich in fermentable carbohydrates Also, hemicellulose rich waste materials like corncob or coconut pith or coconut shell are valuable renewable resources (Tables I and 2). In addition, cultivation of cereals generates a lot of waste residues which is underutilized.

Hydrolysis of Cellulosics

The ultimate product of cellulose hydrolysis is glucose; a well known fermentable six-carbon sugar while the hemicellulosic fraction gives D-xyJpse, a five-carbon sugar. Thus, D-xylose is • the second most abundant sugar in nature and a potential India's production feedstock for generating food and fuel. Cost analysis has indi-· of wheat straw and cated that economically viable bioconversion processes require rice straw is over quantitative conversion of cellulosic as well as herni-cellulosic 200 million tonnes sugars. Hemicellulosic sugars (D-xylose) can be obtained more per year.

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Table 1. Composition of BIOMASS CELLULOSE HEMI- LIGNIN selected Iigno-celluloslc CELLULOSE materials. (% Dry weight - subject to Rice straw 37 24 14 variation.) Wheat straw 31 29 18 Bamboo 40 20 20 Subabul 33 20 NA Mesta wood 35 18 NA

readily with better yield of 80-90% from xylan by acid or enzy matic hy~rolysis than D-glucose from cellulose. Utilization of D-xylose for the commercial production of valuable chemicals like ethanol, acetic acid, 2, 3-butanediol, acetone, isopropanol and n-butanol using microorganisms is thus important for en hancing the economic viability of lignocellulose utilization.

For a long time, it was believed that yeast could not ferment D xylose although many strains can ferment D-xylulose, the keto isomer ofD-xylose. In 1914, a famous microbiologist Kluyver in his PhD thesis mentioned that, " ... only a small amount ofxylose was available. Therefore, I did not routinely include xylose in my fermentation experiments. Although many fungi can assimilate this sugar there are so many reports on their inability to ferment that omission ofxylose was not considered to be serious. The small amounts of xylose available was used in a few experiments with selected yeasts strains. .. all results were negative."

Table 2. Various sugars in • Several yeasts like Candida polymorpha and Pichia muo can agricultural residues. aerobically convert D-xylose to xylitol as the major product. (% Dry weight subJect to varia The conversion efficiency can be as high as 90%. This finding is tion.) also encouraging since xylitol a sugar alcohol, is a natural sweet-

Residue XYLOSE ARABINOSE · GLUCOSE OTHERS

Corn cobs 65 10 25 Wheat straw 58 9 28 3 Flax straw 65 13 1 21 Bagasse 60 15 25 54 * RESONANCE I Mav 2002 GENERAL I ARTICLE ener present in small quantities in a wide variety of fruits and Xylitol does not vegetables. XyIitol does not form acid and may be used clini· form acid and may cally as a sugar substitute for diabetic patients. Xylitol is be used clinically frequently used in chewing gums and toothpaste. Several other as a sugar yeasts are also known to convert D-xylose to xylitol but the substitute for conversion efficiency is only 50%. diabetic patients.

Production of Ethanol from Xylose by Biological Fermentation

The metabolic steps involved in the fermentation of six-carbon sugars have extensively been studied when compared to five carbon sugars. The initial biochemical steps in D-xylose fer· mentation are the isomerization of D-xylose to D-xylulose, the corresponding keto isomer ·(Figure 3). In bacteria, D-xylose isomerase produces D-xylulose from D-xylose. I~ yeast, two enzymatic steps involving reduction and oxidation carry out the same conversion. The NADPH·linked D-xylose reductase con· verts D-xylose to xylitol. In the ne~tep, xylitol is converted to D-xylulose by NAD-linked xylitol dehydrogenase.

0-Xylose kAD(P)H D-Xylose reductase "' [, (Veasts) II! 'j NAD(P} a-Xylose isomerase II Xylitol (Ba'~te ria ;- 1 T7;~~;~~ogenase ~Dti' {Yeasts) D-Xylulose

ADP -- ATP

D-Xylulokinase

D-Xylulose-5-phosphate Figure 3.

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The main problem Phosphorylation of D-xylulose is then catalyzed by D in the use of xylulokinase, which is a chief driving reaction in the pathway_ bacteria is Beyond this point, much of the pathway is assumed from bio· attributed to their chemical studies with other organisms. In one route, D low ethanol xylulokinase proceeds by way of epimerase, isomerase, r tolerance and the transket lase and transaldolase to form glyceraldehyde-3-phos formation of phate and fructose-6-phosphate. Glyceraldehyde-3-phosphate products other is an intermediate, both in the pentose phosphate pathway and than ethanol. the Embden Meyerhof Parnas pathway. The requirement for the induction of these enzymes and their proportion, as well as the availability of the necessary cofactors are thought to be the major factors causing the inefficiency of pentose fermentation.

In the last 20 years, yeasts such as Pachysolen tannophilus, Can dida shehatae and Candida tropicalis have been identified for ethanol production from D-xylose in appreciable yields. Con version of pentose sugars to ethanol in the absence of oxygen is limited to only a small number of yeasts, although many yeasts can assimilate these suga~ aerobically. Some molds such as Fusarium, Rhizopus and Mucor are known to fermentD-xylose to ethanol. Many bacteria are able to utilize xylose; especially members of the genera Clostridium and Bacillus (B. macerans) can carry out mixed fermentation in which ethanol is a major end-product. The main problem in the use of bacteria is attrib uted to their low ethanol tolerance and the formation of prod ucts other than ethanol.

The rates and yields of ethanol from D-xylose depend upon several process variables such as D-xylose concentration, nitro gen source, aeration and agitation. C. shehatae is an efficient fermenter of D-xylose but it is not able to utilize nitrate as the sole nitrogen source. In case of Pachysolen tannophilus, oxygen is required for cell growth but not for ethanol production. P. tannophilus yields 0.34 g ethanol per gram of pentose. D-xylose is assumed to follow the stoichiometry:

3 D-xylose ~ 5 ethanol + 5 CO 2 (1.67 mole of 2-carbon fragment per mole of S-carbon fragment consumed)

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The rate of formation as well as final yields of ethanol from D The fermentation xylose fermentation is still much lower than commercial glucose of D-xylose is fermentation rates. The ethanol production from D-xylose is practicable only lower than the theoretical yields. On mass basis, 0.51 g alcohol where it is a by should be obtained from 1 g of D-xylose. One of the main product of reasons for the low yield is the formation of other fermentation lignocellulose products, such as xylitol, acetate, etc. The improvement in the processing. final yield as well as specific rates of ethanol formation has been achieved by strain improvement through mutation studies. The biochemical basis as well as rate limiting steps during D-xylose utilization by yeasts are being studied in many laboratories including National Chemical Laboratory in Pune. These stud ies may facilitate the design of an appropriately controlled fermentation system and it also serves as a basis for obtaining genetically engineered strains.

All the parameters influencing the fermentation process have not been fully evaluated so far. Variables including aeration, pH of the medium, C:N ratio, various additives and respiratory inhibitors are manipulated in order to achieve high ethanol yields from pentose sugars. It is expected that the production of at least 5-6% (w/v) ethanol will be attained. The fermentation of D-xylose is practicable only where it is a by-product oflignocel lulose processing. Adaptation of yeasts for fermentation using waste streams or hydrolysates is another essential feature neces sary for process development.

Efforts Towards Increasing the Ethanol Yield

We have carried out enzymatic saccharification of various agri cultural residues of Indian origin. For this purpose, an enzyme preparation containing the cellulase/xylanase enzyme complex produced by Sclerotium rolfsii was used. The resulting mixture of the 6-carbon and S-carbon sugars was fermented using Saccharo myces cerevisiae and Candida shehatae, separately and together. The co-culture system gave improved ethanol yields.

We have also carried out D-xylose fermentation in the presence of synthetic aluminosilicates of mineral faujasite structure, zeo-

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The genetically lites of X and Y type or a combination thereof. Incorporation of engineered yeast 1% Na-Y or Na-Ca-X (w/v), in the fermentation medium in produces at least creased the yield of ethanol by 8 to 15%. 30% more ethanol Research on the role that microorganisms play in the from a given ethanologenesis from pentose sugars is still in the developmen amount of plant tal stage. US researchers at Purdue University (Indiana) have materials. developed a genetically modified yeast, which can efficiently ferment cellulose to ethanol, a breakthrough which could slash the cost of production of ethanol from renewable feed stocks. The yeast is effective at cofermenting glucose and xylose from lignocellulosic biomass into ethanol. The genetically engi neered yeast produces at least 30% more ethanol from a given amount of plant materials. The goal is to make ethanol less expensive than gasoline. It is too early to speculate on the commercial significance of the second most abundant sugar available in nature.

Nevertheless, the hydrocarbons of the future need no longer be fossil fuels to be unearthed and processed, but might well be available continuously from our rooftops or gardens. While we wait for the day when we can simulate controlled fusion that Address for Correspondence Anil Lachke generates the energy of the sun and stars, we might also find new Division of Biochemical ways of harnessing solar energy. As Robert Frost said. Sciences National Chemical Laboratory " We dance round in a ring and suppose. Pune 411 008. India. But the secret sits in the middle and knows!"